(a) Field of the Invention
The invention relates to a marker for nuclear restoration of cytoplasmic male sterility, and more particularly to the use of glyceraldehyde-3-phosphate dehydrogenase complementary DNA as such a marker. The invention also relates to a gene for nuclear restoration of cytoplasmic male sterility, and more particularly to the use of a form of the gene encoding glyceraldehyde-3-phosphate dehydrogenase for this purpose. Finally, the invention relates to the production of restorer lines directly through genetic transformation of plants with such a gene.
(b) Description of Prior Art
Hybrids of different crop varieties may show yields that are considerably greater than those of the parental lines. This phenomenon is known as hybrid vigor. To implement the use of hybrid vigor it is necessary to have a method available for preventing self-pollination of one or both of the parent lines in the hybrid cross. Mechanical, chemical and genetic methods are available for accomplishing this. One established genetic method involves the trait of cytoplasmic male sterility (CMS). The genetic determinants for CMS, the maternally transmitted inability to produce viable pollen, reside on the mitochondrial genome. Because CMS plants are male sterile, all of the seed that forms on them will necessarily be hybrid. Due to the maternal transmission of CMS, however, such F1 hybrids will also normally be male-sterile and hence be unable to self-fertilize and produce seed. To address this problem, specific nuclear genes that suppress the male sterile phenotype, termed restorers of fertility (Rf), can be incorporated into the pollinating parent of the hybrid cross. Genotypes on which the male sterile cytoplasm confers sterility are termed maintainers whereas those carrying Rf genes are termed restorers; the genes for the maintenance and restoration of CMS can be considered as different alleles (rf and Rf, respectively) at the same locus.
To produce a diverse set of hybrids using CMS, adequate numbers of restorer lines, that contain Rf genes, as well as xe2x80x9cmaintainerxe2x80x9d lines, that are sterilized by the CMS cytoplasm, must be available. The use of such lines in hybrid crop production is outlined in FIG. 1. The development of these lines through conventional genetics is a slow process that minimally requires several years of effort and currently poses a major bottleneck in the generation of CMS-based hybrids in a number of crops, including canola, Canada""s major cash crop. For example, to create a new restorer line it is necessary to first generate a hybrid between an existing restorer strain, which donates the Rf gene, and a recipient strain; a series of backcrosses to the recipient strain are then performed to incorporate the Rf gene without altering the strain""s other desirable characteristics, a process termed introgression. Even after many generations some donor DNA that is linked to the Rf gene on the donor DNA will remain, a phenomenon termed linkage drag; this donor DNA may carry deleterious traits and compromise the quality of the recipient strain (Jean, M. et al., 1993, Current Topics in Molecular Genetics, 1:195-201).
This process can be expedited through the general process of indirect selection: progeny plants are first screened for genetic markers linked to the restorer gene rather than the restorer gene itself. These markers are chosen such that they can be screened for at a very early stage in plant development. This circumvents the costly procedure of raising many progeny plants to maturity and can considerably accelerate the introgression process. Restriction fragment length polymorphisms (RFLPs) represent a type of DNA marker that is ideally suited for this purpose. RFLPs are differences (between two genotypes) in restriction fragment patterns detected by specific DNA probes. Probes that detect fragment pattern differences between restorer and maintainer lines and that co-segregate with the Rf gene can be used to indirectly select for the restorer gene in a plant breeding program. We have obtained several probes that are linked to Rfp1, a restorer of the Polima or pol CMS, one of the two forms of CMS in canola (B. napus) that is currently being used in hybrid seed production. None of these markers is completely linked to the gene. This introduces an element of uncertainty into their use for indirect selection-the presence of any one marker in a plant does not guarantee the presence of the restorer gene in that plant. It therefore is necessary to employ a number of the markers for indirect selection of plant containing the restorer gene.
It would be highly desirable to be provided with a marker that is perfectly associated with nuclear restoration of cytoplasmic male sterility.
This process can be further expedited through direct introduction of a cloned restorer gene. We believe that the probe we have identified, which show perfect linkage to Rfp1 is detecting the restorer gene itself.
One aim of the present invention is to provide a marker for nuclear restoration associated with cytoplasmic male sterility.
Another aim of the present invention is to provide the use of glyceraldehyde-3-phosphate dehydrogenase complementary DNA as such a restorer marker.
Another aim of the present invention is to be able to use this gene to produce restorer lines directly through genetic transformation.
In accordance with the present invention there is provided a probe specific for nuclear restoration of cytoplasmic male sterility of plants, which comprises a glyceraldehyde-3-phosphate dehydrogenase cDNA or genomic DNA sequence, a hybridizing fragment thereof or any DNA sequence derived therefrom for use as primers for amplification of glyceraldehyde-3-phosphate dehydrogenase, wherein said DNA sequence or hybridizing fragment thereof hybridizes to specific DNA fragments characteristic of plants possessing a nuclear restorer gene under stringent conditions.
In accordance with the present invention there is also provided a gene for nuclear restoration of cytoplasmic male sterility in plants which comprises a DNA sequence encoding glyceraldehyde-3-phosphate dehydrogenase and surrounding sequences.
The surrounding sequences may be located 3xe2x80x2 and/or 5xe2x80x2 relative to the glyceraldehyde-3-phosphate dehydrogenase sequence and may be of about 50 kb.
In accordance with the present invention there is also provided a method of production of restorer lines, which comprises genetically transforming plants with the nuclear restoration of cytoplasmic male sterility gene of the present invention.
In accordance with the present invention, any plant species may be used provided that the restorer gene in the plant species corresponds to a specific form of GAPC. Such species include, without limitation, Brassica napus, other Brassica species, maize (Zea mays), rice (Oryza sativum), sunflower (Helianthus annuum) and sorghum (Sorghum bicolor).